Recombinant Bat coronavirus HKU9 Envelope small membrane protein (E)

What is the basic structure of Bat coronavirus HKU9 Envelope small membrane protein (E)?

The Bat coronavirus HKU9 Envelope small membrane protein (E) consists of 79 amino acids with a sequence of: MYDIVGTNNSILIANVLVLIIICLLVVIVGCALLLILQFVFGVCGFVFKFVCKPTILVYNKFRNESLLNEREELLCDNV . The E protein is a small transmembrane protein that plays critical roles in coronavirus assembly, budding, and pathogenesis. Structurally, it contains hydrophobic transmembrane domains that anchor the protein within the viral envelope and hydrophilic regions that interact with other viral and host proteins. This architecture is consistent with other coronavirus E proteins that typically form viroporins (ion channels) in the viral membrane.

What are the optimal expression systems for producing recombinant Bat coronavirus HKU9 E protein?

Based on research protocols, three expression systems have been successfully employed for producing recombinant Bat coronavirus HKU9 E protein:

For purification, affinity chromatography using His-tagged proteins followed by size exclusion chromatography yields highly pure protein suitable for structural and functional studies.

What are the critical considerations for maintaining protein stability and functionality during purification?

Maintaining stability and functionality of the Bat coronavirus HKU9 E protein requires specific buffer conditions and handling protocols:

• Buffer Optimization: Typically stored in Tris-based buffers (pH 8.0-8.5) with 300 mM NaCl to maintain ionic strength and 10-50% glycerol as a stabilizing agent .

• Lyophilization Protocol: For long-term storage, the protein can be lyophilized from a solution containing protectants such as 5-8% trehalose, mannitol, and 0.01% Tween80 .

• Storage Recommendations: Aliquot and store at -20°C to -80°C to avoid repeated freeze-thaw cycles which can cause protein degradation . For working aliquots, storage at 4°C is limited to one week .

• Reconstitution Method: Centrifuge the vial before opening to recover the entire contents, and reconstitute in sterile water to prepare a stock solution (typically 0.2 mg/ml) .

How does the Bat coronavirus HKU9 utilize host cell receptors compared to SARS-CoV and MERS-CoV?

Detailed research using surface plasmon resonance (SPR) has demonstrated significant differences in receptor utilization between Bat coronavirus HKU9 and other betacoronaviruses:

• Receptor Binding Domain (RBD) Characteristics: The HKU9-RBD is composed of a core subdomain and an external subdomain. The core subdomain fold resembles those of other betacoronavirus RBDs, whereas the external subdomain is structurally unique with a single helix rather than the beta-sheet topology observed in other betacoronaviruses .

• Receptor Affinity Testing: SPR analysis revealed that HKU9-RBD binds to neither the SARS-CoV receptor ACE2 nor the MERS-CoV receptor CD26 (also known as DPP4) . This was confirmed using both insect cell-expressed and mammalian cell-expressed proteins to exclude any effects of improper post-translational modifications.

• Binding Kinetics: While SARS-RBD–ACE2 shows binding with KD = 0.265 μM and MERS-RBD–CD26 with KD = 52.8 nM, no detectable binding was observed between HKU9-RBD and either receptor under identical experimental conditions .

• GRP78 Interaction: Research has identified that HKU9 can utilize GRP78 (a heat shock protein) for attachment to host cell surfaces, representing an alternative mechanism for viral attachment . This interaction between GRP78 and spike proteins occurs in both MERS-CoV (lineage C betacoronavirus) and HKU9 (lineage D betacoronavirus), suggesting a conserved alternative attachment strategy.

These findings indicate that Bat coronavirus HKU9 employs a distinct receptor recognition mechanism from SARS-CoV and MERS-CoV, highlighting the diverse evolution of receptor usage within betacoronaviruses.

What role does GRP78 play in Bat coronavirus HKU9 attachment to host cells?

Recent research has revealed that GRP78 (78 kDa glucose-regulated protein), a cellular chaperone protein, can interact with the spike protein of Bat coronavirus HKU9 and facilitate its attachment to host cell surfaces :

• GRP78-Spike Interaction: Experimental evidence demonstrates that the highly conserved human GRP78 can interact with the spike protein of bCoV-HKU9 and facilitate its attachment to the host cell surface.

• Evolutionary Significance: This capacity of GRP78 to facilitate surface attachment of both a human coronavirus (MERS-CoV) and a phylogenetically related bat coronavirus (HKU9) exemplifies potential mechanisms for cross-species transmission.

• Mechanism Distinction: The GRP78 interaction represents an attachment factor rather than a primary entry receptor, potentially augmenting virus attachment to host cells through a conserved mechanism.

• Implications for Surveillance: The identification of this shared attachment mechanism between human and bat coronaviruses underscores the importance of monitoring the evolution of animal coronaviruses for potential human adaptation pathways.

This research highlights alternative viral attachment strategies that might play roles in cross-species transmission and host range determination for coronaviruses.

What evolutionary relationships exist between Bat coronavirus HKU9 and other coronaviruses?

Bat coronavirus HKU9 occupies a distinct evolutionary position within the betacoronavirus genus:

• Taxonomic Classification: BatCoV HKU9 belongs to subgroup D of betacoronaviruses, representing a lineage distinct from SARS-CoV (subgroup B), MERS-CoV (subgroup C), and human coronaviruses like HKU1 (subgroup A) .

• Phylogenetic Analysis: Phylogenetic trees constructed using multiple viral proteins (including chymotrypsin-like protease, RNA-dependent RNA polymerase, helicase, spike, and nucleocapsid) consistently show that group 2a, 2b, 2c, and 2d coronaviruses are more closely related to each other than to group 1 and 3 coronaviruses .

• Genomic Features: Unique genomic characteristics distinguish between these four betacoronavirus subgroups, including the number of papain-like proteases, the presence or absence of hemagglutinin esterase, small ORFs between membrane and nucleocapsid genes, and specialized RNA structures .

• Evolutionary Divergence: The receptor binding domains (RBDs) of these viruses show particular evolutionary divergence, with HKU9-RBD having a structurally unique external subdomain compared to other betacoronaviruses, explaining its inability to use ACE2 or CD26 as receptors .

This evolutionary context helps researchers understand the potential for host-switching events and the emergence of novel coronaviruses with pandemic potential.

How do genetic recombination events shape Bat coronavirus HKU9 evolution?

Genetic recombination plays a crucial role in coronavirus evolution, with several significant findings regarding Bat coronavirus HKU9:

• Coexisting Genotypes: Research has demonstrated the remarkable phenomenon of different Bat coronavirus HKU9 genotypes coexisting within the same bat host. Among 10 bats with complete RNA-dependent RNA polymerase (RdRp), spike (S), and nucleocapsid (N) genes sequenced, multiple sequence clades were codetected in the same individual bats .

• Complete Genome Evidence: Complete genome sequencing of distinct genotypes from individual bats confirmed the coexistence of at least two distinct genomes in each bat, representing the first report describing coinfection of different coronavirus genotypes in bats .

• Recombination Mechanisms: Recombination analysis using multiple Bat coronavirus HKU9 genomes revealed evidence of recombination events between strains from different bat individuals, which may facilitate the generation of different genotypes .

• Cross-Family Recombination: In related bat coronaviruses (such as Ro-BatCoV GCCDC1, which also belongs to betacoronavirus group D), researchers have identified evidence of potential inter-family recombination between coronaviruses and orthoreoviruses, suggesting that heterologous recombination may contribute to coronavirus evolution .

• Ecological Factors: The dense roosting behavior and long foraging range of fruit bats like Leschenault's rousette may create opportunities for viral coinfection and recombination .

These findings highlight the complex evolutionary dynamics of bat coronaviruses and their potential for generating genetic diversity through recombination.

What crystallographic methods have been successful in determining Bat coronavirus HKU9 protein structures?

The structural determination of Bat coronavirus HKU9 proteins, particularly the receptor binding domain (RBD), has been accomplished using specific crystallographic techniques:

• Crystallization Conditions: The crystallization of HKU9-RBD was achieved using the vapor-diffusion sitting-drop method at 4°C, with optimal crystals obtained under conditions of 0.1 M sodium citrate tribasic dihydrate (pH 7.0) and 12% PEG 20000 with a protein concentration of 2.2 mg/mL .

• Derivative Preparation: Heavy atom derivatives were prepared by soaking crystals in reservoir solution containing 1 mM KAuBr₄·2H₂O for 48 hours at 4°C .

• Data Collection Parameters: The crystallographic data collection used the following parameters:

|Parameter|HKU9-RBD (PDB: 5GYQ)|Au derivative HKU9-RBD|
|--|--|
|Space group|P21|P1|
|Wavelength (Å)|1.03906|1.03906|
|Unit cell dimensions|||
|a, b, c (Å)|42.7, 36.0, 62.9|36.0, 46.6, 57.3|
|α, β, γ (deg)|90.0, 102.7, 90.0|80.4, 88.8, 88.5|
|Resolution (Å)|50.00–2.10 (2.18–2.10)|50.00–2.48 (2.57–2.48)|
|Observations|101588|52401|
|Completeness (%)|97.1 (80.7)|97.7 (96.9)|

• Refinement Statistics: The structure was refined to Rwork/Rfree values of 0.1700/0.2006, with Ramachandran statistics showing 94.64% of residues in favored regions and 5.36% in allowed regions, with no outliers .

• Structural Resolution: The final structure contained a single molecule in the crystallographic asymmetric unit with clear electron densities traced for 176 consecutive HKU9-RBD residues (S355 to A520), revealing the two-subdomain architecture of the protein .

These methodological details provide crucial guidance for researchers seeking to determine structures of other coronavirus proteins.

What techniques are effective for studying Bat coronavirus HKU9 E protein interactions with host factors?

Multiple complementary techniques have proven effective for investigating Bat coronavirus HKU9 E protein interactions:

• Surface Plasmon Resonance (SPR):

  • Direct SPR: Immobilizing the E protein on a sensor chip surface and flowing potential host proteins over it to detect binding.

  • Captured SPR: For fusion-tagged proteins, anti-tag antibodies can be immobilized on the sensor chip to capture the protein of interest in a defined orientation.

  • Experimental parameters: Regeneration of chip surfaces typically uses 10 mM glycine (pH 1.7) or 10 mM NaOH .

• Co-immunoprecipitation Assays:

  • Expressing tagged versions of the E protein in mammalian cells.

  • Using anti-tag antibodies to pull down protein complexes.

  • Identifying interacting partners through Western blotting or mass spectrometry.

• Serological Characterization:

  • Western blot assays using recombinant E proteins have demonstrated subgroup-specific antibody responses.

  • Enzyme immunoassays show high seroprevalence (43-64%) in bat populations, enabling epidemiological studies .

• Biochemical Characterization:

  • Circular dichroism spectroscopy to assess secondary structure.

  • Size exclusion chromatography to determine oligomeric state.

  • Lipid binding assays to investigate membrane interactions.

• Reverse Genetics Systems:

  • Engineering mutations or deletions in the E protein gene.

  • Assessing the impact on viral replication, assembly, and release.

  • This approach requires establishing infectious clone systems, which remain challenging for many bat coronaviruses.

These methods provide complementary approaches to understand both the structural properties and functional roles of the E protein in coronavirus biology.

How might Bat coronavirus HKU9 E protein contribute to understanding zoonotic potential?

The study of Bat coronavirus HKU9 E protein offers several insights into zoonotic potential:

• Comparative Evolutionary Analysis: Comparing the E protein sequences and structures between bat and human coronaviruses can reveal conserved functional domains versus host-specific adaptations. The conservation of certain E protein motifs across diverse coronaviruses may indicate functional constraints that limit evolutionary divergence, even during host-switching events.

• Pathogenesis Mechanisms: The E protein plays critical roles in virion assembly, release, and pathogenesis. Research on SARS-CoV and other coronaviruses has shown that the E protein contributes to viral pathogenicity through ion channel activity and interactions with host proteins involved in inflammatory responses.

• GRP78 Interaction Pathway: The discovery that both MERS-CoV and Bat coronavirus HKU9 can utilize GRP78 for attachment despite belonging to different betacoronavirus lineages suggests a conserved mechanism that may facilitate cross-species transmission . This highlights a potential pathway for bat coronaviruses to adapt to human cells without requiring specific mutations in the receptor binding domain.

• Recombination Potential: The documented coexistence of multiple Bat coronavirus HKU9 genotypes within single bat hosts provides opportunities for recombination events that could generate viruses with novel properties . The identification of potential inter-family recombination between coronaviruses and orthoreoviruses further underscores the genomic plasticity that may contribute to emergence of viruses with altered host ranges .

• Serological Evidence: High seroprevalence (64%) in tested bat populations indicates widespread circulation of these viruses, providing ample opportunity for evolution and adaptation .

Understanding these aspects of the E protein biology may help predict which bat coronaviruses possess greater potential for cross-species transmission and inform surveillance strategies targeting high-risk viral lineages.

What role does the Bat coronavirus HKU9 E protein play in viral assembly and pathogenesis?

The E protein of Bat coronavirus HKU9, like other coronavirus E proteins, likely plays multiple crucial roles in the viral life cycle:

• Virion Assembly and Morphogenesis: The E protein is essential for proper virion assembly and morphogenesis in coronaviruses. It interacts with other structural proteins (particularly M protein) to drive the budding process and determine virion shape.

• Ion Channel Activity: Coronavirus E proteins form viroporins (ion channels) in the lipid bilayer. This activity may alter host cell membrane permeability and ion homeostasis, potentially contributing to pathogenesis through effects on the secretory pathway and inflammatory responses.

• Protein-Protein Interactions: The E protein's C-terminal domain likely engages in specific interactions with host cell proteins. In other coronaviruses, such interactions have been shown to modulate host cell signaling pathways, including those involved in inflammation and stress responses.

• Viral Release: The E protein facilitates efficient release of viral particles from infected cells. Studies with other coronaviruses have shown that deletion or mutation of the E protein can result in attenuated viruses with defects in virion release.

• SARS-Unique Fold Domain Interactions: Research on the related C domain of Bat coronavirus HKU9 (a domain within nsp3) has revealed structural similarities to SARS-CoV despite low sequence identity . This suggests conserved functional roles across betacoronaviruses that may involve interactions with viral or host factors during replication.

Although direct experimental evidence for these functions specifically in Bat coronavirus HKU9 remains limited, comparative analysis with other coronavirus E proteins suggests conserved mechanisms. Further research using reverse genetics and biochemical approaches would be necessary to definitively characterize these functions in the context of Bat coronavirus HKU9 infection.

What are the key unresolved questions regarding Bat coronavirus HKU9 E protein?

Despite advances in understanding the Bat coronavirus HKU9 E protein, several critical questions remain unresolved:

• Receptor Identification: While we know that HKU9 RBD doesn't bind to ACE2 or CD26, the primary cellular receptor for HKU9 remains unidentified. Beyond the GRP78 attachment factor, what is the primary entry receptor for this virus?

• Structure-Function Relationships: The high-resolution structure of the HKU9 E protein remains undetermined. How does its structure relate to its various functions in the viral life cycle?

• Ion Channel Properties: Does the HKU9 E protein form functional ion channels like other coronavirus E proteins? If so, what are its ion selectivity and conductance properties?

• Host Range Determinants: What molecular features of the E protein contribute to host specificity? Are there specific mutations that could expand its host range?

• Immune Evasion Mechanisms: How does the E protein interact with the host immune system? Does it play roles in antagonizing innate immune responses?

• Role in Viral Fitness: How does the E protein contribute to viral replication efficiency and transmission in natural bat hosts?

• Comparative Virology: How do the functions of the HKU9 E protein compare with those of other betacoronaviruses, particularly those with zoonotic potential?

• Post-Translational Modifications: What post-translational modifications occur on the native E protein, and how do they regulate its functions?

Addressing these questions will require a combination of structural biology, reverse genetics, biochemical assays, and in vivo models.

What novel experimental approaches could advance understanding of Bat coronavirus HKU9 E protein?

Innovative experimental approaches could significantly enhance our understanding of Bat coronavirus HKU9 E protein:

• Cryo-Electron Microscopy: High-resolution structural determination of the E protein in its native membrane environment could reveal oligomerization states and ion channel conformations.

• Single-Molecule Techniques: Applying single-molecule fluorescence or force spectroscopy to study the dynamics of E protein interactions with host factors or other viral proteins.

• Advanced Cell Models: Development of bat cell lines or organoids that more closely mimic the natural host environment could provide insights into species-specific functions.

• In Situ Structural Biology: Techniques like cryo-electron tomography could visualize the E protein within virions or infected cells, providing context for its structural arrangements.

• Synthetic Biology Approaches: Creating chimeric viruses or E proteins to map functional domains and host range determinants.

• Systems Biology Integration: Multi-omics approaches combining proteomics, lipidomics, and transcriptomics to comprehensively map E protein interactions and effects.

• Advanced Computational Modeling: Molecular dynamics simulations of the E protein in membranes could predict conformational changes and ion conduction mechanisms.

• CRISPR-Based Screening: Genome-wide screens to identify host factors that interact with or are modulated by the E protein.

• Reverse Genetics Systems: Development of infectious clones or replicon systems for Bat coronavirus HKU9 would enable direct manipulation of the E protein in the viral context.

• Interdisciplinary Collaboration: Combining virology with biophysics, structural biology, and evolutionary biology could provide more comprehensive insights into E protein function and evolution.

These approaches would complement existing research methods and potentially overcome current limitations in studying this challenging but important viral protein.